animal models in lung research

1. Animal Models in Lung Research David Aronoff, MD Asst. Professor Division of Infectious Diseases Department of Internal Medicine Graduate Program in ImmunologyProgram in Biomedical Science daronoff@umich.edu

3. Outline The diseases that are modeled in animals Mice: pros & cons Measuring lung function in mice Models of: Pulmonary fibrosis Asthma Bacterial pneumonia Novel imaging tools Conclusions

4. Pulmonary diseases commonly modeled in animals Asthma Cancer COPD Pneumonia Pulmonary hypertension Interstitial lung disease (pulmonary fibrosis)

5. The mouse model Mice are commonly used to model lung disease This talk will focus on mouse models

6. Using mice for research Advantages Small size & rapid reproductive rate Genotypically homogeneous Inbred strains allow the study of identical cohorts Facilitate genetic approaches to understanding molecular mechanisms of disease Genetic engineering of mouse embryonic stem cells allows Generation of mice with loss-of-function, gain-of function, reporter genes in the genome Allows cell specific or condition-specific studies Mouse reagents readily available

7. Using mice for research Disadvantages Differences among mouse strains can be major It has been advocated to use > 1 strain per study to control for this Some diseases are different in mouse & man Asthma models Fibrosis models

8. The big concern: a mouse is not a human

9. Measuring lung function in rodents Key reference: Hoymann HG. Invasive and noninvasive lung function measurements in rodents. J Pharmacol Toxicol Methods. 2007 Jan-Feb;55(1):16-26.

10. Measuring lung function in rodents: key principles The principles governing ventilation, air flow, lung volume, & gas exchange are shared among mammals The functional responses of man & other animals to different types of lung injury are similar

11. Measuring lung function in rodents Existing methods for measuring respiratory function in rodents in vivo include both invasive & noninvasive approaches ��there is no way to measure lung function in mice both accurately & noninvasively.� Lundblad LKA, et al. J App Physiol 2003

12. Measuring lung function in rodents

13. Noninvasive techniques Noninvasive (PRO) Convenient Repeatable Simple to handle Screen many animals/day Nearly natural breathing pattern

14. Noninvasive techniques Noninvasive (CON) Stress Only volume & flow measurements No resistance or compliance data Inhalation exposure includes nasal & GI uptake

15. Measuring lung function in rodents

16. Invasive techniques Invasive (PRO) No stress Gold standard parameters (resistance, compliance, diffusion) Inhalation exposure focused to lungs

17. Invasive techniques Invasive (CON) Takes more time Not easy to do Anesthesia depresses respiration

18. Pulmonary fibrosis Key references: Moore BB & Hogaboam CM. Am J Physiol Lung Cell Mol Physiol 294:152-160, 2008.

19. Pulmonary fibrosis IPF is characterized by Alveolar epithelial cell injury & hyperplasia Inflammatory cell accumulation Fibroblast hyperplasia Deposition of extracellular matrix Scar formation End result is loss of elasticity & alveolar surface area ? impairment of gas exchange & pulmonary function

20. Pulmonary fibrosis in mice Methods to induce fibrosis in mice: BLEOMYCIN FLUORESCEIN ISOTHIOCYANATE (FITC) IRRADIATION SILICA TRANSGENE EXPRESSION VIRAL VECTOR INFECTION ADOPTIVE TRANSFER OF HUMAN FIBROBLASTS INTO IMMUNODEFICIENT MICE

21. Pulmonary fibrosis in mice Bleomycin (a chemo drug) Best characterized mouse model in use Causes pulmonary fibrosis in humans too i.v., i.p., s.q., & inhaled routes all cause fibrosis The i.v. model approximates the pathogenesis of human bleo-induced fibrosis

22. Pulmonary fibrosis in mice Bleomycin cont� Following i.v. administration initial lesions involve the pulmonary endothelium Acute lung injury Damage to the alveolar epithelium Leakage of fluid & plasma proteins into the alveolar space Alveolar consolidation & the formation of hyaline membranes

23. Pulmonary fibrosis in mice Bleomycin cont� Focal necrosis of type I & metaplasia of type II AECs Inflammatory infiltrates Subpleural fibrosis Collagen accumulation Measured by histological & biochemical means Lung hydroxyproline is a surrogate for collagen

24. Pulmonary fibrosis in mice Bleomycin cont� Disadvantages: Fibrosis does not develop in all animals The time frame for the development of fibrosis is relatively long 1st observed at 4 wk & more severe by 12 wk (faster with i.t.) The fibrotic response is strain-dependent C57Bl/6 more susceptible than BALB/c mice

25. Pulmonary fibrosis in mice

26. Pulmonary fibrosis in mice Example of bleo model in use Comparison of fibrosis in mice lacking the gene encoding 5-lipoxygenase

27. Pulmonary fibrosis in mice FITC i.t. administration of FITC to BALB/c & C57Bl/6 mice By 5 mo post-FITC, patchy, focal destruction of normal lung architecture + interstitial fibrosis

28. Pulmonary fibrosis in mice Fibrosis develops in areas of FITC deposition Day 21 after i.t. FITC A: H&E: consolidation, inflammation & fibrosis B: UV light shows FITC deposition Areas of fibrosis correspond to areas of FITC Red arrows = normal architecture, where FITC did not deposit.

29. Pulmonary fibrosis in mice FITC cont� Advantages: Visualize areas where deposition occurs via immunofluorescence Response is persistent (for at least 6 mo) & not self-limiting like bleomycin Disadvantages: Response varies with the �lot #� of FITC Model not as clinically relevant as bleomycin

30. Pulmonary fibrosis in mice FITC cont� Evaluation of the effect of g herpes virus infection on pulmonary fibrosis

31. Asthma Key references: Nials AT & Uddin S. Dis Model Mech. 2008 Nov�Dec; 1(4-5): 213�220 Brown RH, et al. Proc Am Thorac Soc. 2008, 5:591-600 Zosky GR & Sly PD. Clin Exp Allergy. 2007 Jul;37(7):973-88 Kumar RK & Foster PS. Am J Respir Cell Mol Biol. 2002 Sep;27(3):267-72

32. Asthma Human allergic asthma is a chronic inflammatory disorder Characterized by Inflammation Persistent airway hyperresponsiveness (AHR) Intermittent, reversible airway obstruction Airway remodeling Subepithelial & airway wall fibrosis Goblet cell hyperplasia/metaplasia Smooth muscle thickening Increased vascularity

33. Asthma in mice Mice do not spontaneously get asthma Artificial asthmatic-like reactions are induced Models of acute allergic response to inhaled allergens widely used The nature of the acute inflammatory response influenced by: The mouse strain The allergen The sensitization & challenge protocol

34. Asthma in mice The BALB/c mouse is common Robust Th2 type immune response Chicken ovalbumin (OVA) is a frequent allergen OVA seldom implicated in human asthma Interest in house dust mite (HDM) & cockroach antigen

35. Asthma in mice Acute sensitization protocols often use multiple systemic administrations of the allergen + an adjuvant Aluminum hydroxide (AlOH3) ? Th2 response Short-term exposure to a high mass concentration of allergen ? the recurrent long-term exposure to allergen seen in human disease

36. Asthma in mice

37. Asthma in mice

38. Features of mouse asthma Similarities with human disease: Elevated IgE levels Airway inflammation Goblet cell hyperplasia Epithelial hypertrophy AHR to specific stimuli In some models, early- & late-phase bronchoconstriction in response to allergen challenge

39. Features of mouse asthma Differences from human disease: Pattern & distribution of pulmonary inflammation The intra-epithelial accumulation of eosinophils seen in humans is absent in the mouse model No chronic inflammation of the airway wall No airway remodeling: Subepithelial fibrosis Epithelial proliferation

40. Asthma in mice Acute asthma models have characterized inflammatory mediators, but� Therapeutic targets that work in these models sometimes fail in human clinical trials: Antagonists for IL-5, VLA-4, PAF, & IL-4

41. Asthma in mice Chronic allergen challenge models: Attempt to model chronic AHR & remodeling Repeated airway exposure to low allergen levels up to 12 wks Reproduce some hallmarks of human asthma including allergen-dependent sensitization Might provide a more suitable system for the preclinical evaluation of novel therapeutic agents

42. Asthma in mice

43. Asthma in mice: chronic allergen exposure Similarities with human asthma: A Th2-dependent allergic inflammation Eosinophilic influx into the airway mucosa AHR Airway remodeling: Goblet cell hyperplasia Subepithelial fibrosis Epithelial hypertrophy

44. Asthma in mice: chronic allergen exposure Differences from human asthma: Inflammation not limited to conducting airways (seen in parenchyma & perivascular regions) No large increases in airway smooth muscle Little to no mast cell involvement

45. Modeling pneumonia Reference: Mizgerd JP & Skerret SJ. Am J Physiol Lung Cell Mol Physiol 294: L387�L398, 2008

46. Modeling pneumonia in mice Two practical considerations for studying pneumonia in mice: HOW TO GET MICROBES INTO THE LUNGS HOW TO GET INFORMATION OUT

47. Modeling pneumonia in mice Getting microbes into the lungs: Exposure to infected animals Cohousing uninfected animals with inoculated seed animals Mimics natural infection BUT: inability to control inoculum, time of infection, or the number of infected animals Exposure to aerosolized microorganisms Exposure to aerosolized microorganisms in whole-body or nose-only chambers by nebulization Whole-body chambers coat the whole mouse with pathogen Some bugs do not survive dessication/nebulization well

48. Modeling pneumonia in mice Getting microbes into the lungs (continued): Direct endotracheal or endobronchial instillation Microbial suspension injected in a volume of 5�50 ml Permits precise dosing to the lungs of individual animals Biocontainment is simpler Equipment less costly than with aerosol exposures Anesthesia is required Caveat: the inoculum is deposited predominantly in the lower lung zones in a nonuniform manner Can lead to sampling errors for tissue measurements

49. Modeling pneumonia in mice Getting microbes into the lungs (continued): Intranasal instillation Popular & SIMPLE Anesthetized mice held vertically Microbial suspension deposited in the nares, then aspirated into lungs Major limitation: highly variable deposition of bugs, with inter-animal differences in deposition often > 10-fold Strain differences in snout anatomy can alter results May result in co-aspiration of upper respiratory flora & microbial products that can elicit confounding host responses

50. Modeling pneumonia in mice Getting the information out of the mice: Measuring the microbial burden in mouse lungs (blood, spleen, etc) Standard = quantitative cultures Limited by single time point per mouse Mice are sacrificed to get data Location of bacteria in lungs hard to determine w/ homogenates Detection of nucleic acids by real-time PCR Good for fastidious bugs (think Pneumocystis) Less cumbersome than culture-based modes Does not differentiate live from dead bugs

51. Modeling pneumonia in mice Getting the information out of the mice: Measuring the microbial burden in mouse lungs (blood, spleen, etc) continued� Bioluminescence Based on detection of light produced by microorganisms engineered to express a luciferase gene & its specific substrate Allows animals to be monitored alive, over time Can track the location of infection Limitations: need for luciferase-expressing pathogen; costly imaging system; the limited sensitivity & resolution of current technology

52. Modeling pneumonia in mice

53. Modeling pneumonia in mice Getting the information out of the mice (continued): Measuring the inflammatory response: Neutrophil recruitment Enumeration in BAL fluid (flow cytometry too) Histology Myeloperoxidase measurements Pulmonary edema formation Wet-to-dry weight ratios BALF protein concentrations Microscopy IV injected tracers (such as dyes, etc.)

54. Modeling pneumonia in mice Getting the information out of the mice (continued): Measuring sequelae of inflammation: Changes in lung mechanics Arterial blood gases Bioluminescent surveillance of host gene induction X-rays, PET scans, CT scans, MRIs Serum/organ inflammatory mediator measurements ELISA PCR Western Blot

55. On the horizon Novel small-animal imaging applications Reference: Brown RH, et al. An official ATS conference proceedings: advances in small-animal imaging application to lung pathophysiology. Proc Am Thorac Soc. 2008, 5:591-600 Videomicroscopy MRI Micro-CT Micro-PET Molecular markers

56. On the horizon Videomicroscopy To study dynamic alveolar mechanics during ventilation in real-time with a living animal

57. On the horizon MRI Using �hyperpolarized� gases like 3He & 129Xe to achieve very high resolution of lung, air, & blood structures

58. On the horizon Micro-CT Respiratory gating to image animals at near full inspiration Improves visible lung tissue contrast & detects pathology Can accurately measure tumor volumes Radiation exposure is a limitation

59. On the horizon Micro-PET Coupled w/ CT imaging Noninvasive metabolic information about in vivo processes Used to assess tumor growth

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61. Conclusions Mouse models of lung diseases are popular Many are informative All have important caveats Inter-strain variability can be tackled by comparing models of disease among different mouse strains Many investigators here at the U of M are using mouse models of disease

62. Contact information David Aronoff, MD 4618-C Med Sci Bldg II daronoff@umich.edu 734-647-1786